DE69424184T2 - Method of making peptide products - Google Patents

Abstract

A method in which an esterification reaction is carried out on at least one esterifiable grouping of a protein or peptide contained in an animal or vegetable substrate, and said protein or peptide is exposed to a proteolytic enzyme. The esterification reaction is preferably carried out on at least one carboxylic grouping of the protein or peptide, advantageously by means of an aliphatic alcohol having 1-5 carbon atoms, or an activated derivative thereof. The method may be used to prepare novel peptide populations for use as ingredients or additives in food compositions, pharmaceuticals or cosmetics.

Description

The present invention relates to a process for obtaining peptide products from a substrate of animal or plant origin containing at least one protein or one peptide, as defined in the claims.

According to the present description, the term "peptide products" defines very different peptidic populations, which may contain different entities such as peptides, peptidic fractions, esterified and non-esterified amino acids and their mixtures.

Proteins of animal or plant origin and the peptides derived from these proteins are widely used in all types of industries (pharmaceutical, chemical, food, cosmetics, etc.) due to their chemical or physico-chemical properties.

Enzymatic hydrolysis of milk proteins produces peptides with different biological properties. The same applies to the opium peptides (Zioudrou C., Streaty R. A. and Klee W. A. (1979), J. Biol. Chem. 254, 2446-2449; Brantl V., Teschemacher H., Henschen A. and Lottspeich F. (1981) Life Sci. 28, 1903-1909; Yoshikawa M., Yoshimura T. and Chiba H. (1984), Agric. Biol. Chem. 48, 3185-3187; Loukas S., Varoucha D., Zioudrou C., Streaty R. A. and Klee W. A. (1983), Biochemistry 22, 4567-4573; Chiba H., Tani F. and Yoshikawa M. (1989), J. Dairy Res. 56, 363- 366; Fukudome S. I. and Yoshikawa M. (1992), FEBS Lett. 296, 107-111;

Antila P., Paakkari I., Järvinen A., Mattila M. J., Laukkanen M., Pihlanto-Leppälä A., Mäntsälä P. and Hellman J. (1991) Int. Dairy Journal 1, 215-229; Meisel H. and Frister H. (1989), J. Dairy Res. 56, 343-349).

In addition, antihypertensive peptides (Maruyama S., Mitachi H., Tanaka H., Tomizuka N. and Suzuki H. (1987) Agric. Biol. Chem. 51, 1581-1586; Komura M., Nio N., Kubo K., Minoshima Y., Munekata E. and Ariyoshi Y. (1989), Agric. Biol. Chem. 53, 2107-2114) and immunostimulatory peptides (Berthou J., Migliore-Samour D., Lifchitz A., Delettré J., Floc'h F. and Jollès P. (1987), FEBS Lett 218(1), 55-58; Migliore Samour D., Floc'h F. and Jollès P. (1989) J. Dairy Res. 56, 357-362) or antithrombotic peptides (Fiat A. M., Levy-Toledano S. P., Caen J. and Jollès P. (1989), J. Dairy Res. 56, 351-355).

On the other hand, the peptides derived from milk proteins have equally interesting physicochemical and interfacial properties (Fox P. F. and Mulvihill D. M. (1983), in Proceedings of IDF Symposium, Helsingør, Denmark, 188-259; Chobert J. M., Bertrand-Harb C. and Nicolas M. G. (1988a), J. Agric. Food Chem. 36, 883-892; Chobert J. M., Sitohy, M. Z. and Whitaker, J. R. (1988b), J. Agric. Food Chem. 36, 220-224; Chobert J. M., Bertrand-Harb C., Dalgalarrondo M. and Nicolas M. G. (1989a), J. Food Biochem. 13, 335-352; Chobert J. M., Touati A., Bertrand-Harb C., Dalgalarrondo M. and Nicolas M. G. (1989b), J. Food Biochem. 13, 457-473; Vuillemard J. C., Gauthier S. and Paquin P. (1989 Lait 69, 323-351; Turgeon L. S., Gauthier F. S., Molle D. and Leonil J. (1992), J. Agric. Food Chem. 40, 669-675).

From a nutritional and therapeutic point of view, enzymatic hydrolysis allows to reduce the allergenicity of serum proteins (Jost R., Monti J. CV. and Pahud J. J. (1987) Food Technol. 41, 118-121).

As a bibliographically relevant reference, one can also cite the patent EP-22019, which describes an enzymatic total hydrolysate of lactose serum proteins and applications of this hydrolysate as a medicine and in therapeutic nutrition.

From a physiological point of view, the hydrolysates of proteins of lactose serum have the property of stimulating the growth of skin regeneration (Wenner V. (1982), Lait 62, 549-565).

It is known to act on proteins or peptides of animal or plant origin and to modify their properties, in particular by grafting substituents.

In particular, document WO-A-9318180 describes the esterification of peptides of animal or plant origin by a process called "enzymatic synthesis of alkyl esters of peptides" and requiring the use of enzymes having both proteolytic and esterolytic activity. The process in question is characterized by the simultaneous contact of the protein substrate with the enzyme and with an alcohol, which leads to the formation of alkyl esters of peptides.

On the other hand, document EP-A-0 047 879 describes a process for preparing protein compositions modified by the addition of one or more amino acids. This process consists in hydrolyzing a protein substrate by an enzyme, adding the additional amino acid(s) which have been esterified in advance, and finally allowing the enzyme to act in such a way as to allow the reconstitution of the protein with the additional amino acid(s).

The aim of the present invention is a process which consists in esterifying a protein substance of animal or vegetable origin which contains at least one protein or one peptide and then enzymatically hydrolysing the esterified protein substance, the process leading to a new peptide population.

The modification of proteins by esterification leads to a new family of peptides with novel physicochemical and biological properties.

The process according to the invention consists in carrying out an esterification reaction with at least one esterifiable group of at least one of the proteins or peptides contained in the substrate of animal or plant origin in order to modify the subsequent action of the enzyme (cleavage sites and reaction kinetics).

Non-limiting examples of protein or peptide substrates that can be used include: milk proteins (caseins and lactose serum proteins), wheat or corn gluten, zein, soya, pea and broad bean protein concentrates and isolates, or collagen, serum albumin, ovalbumin, etc.

This esterification reaction is carried out according to the classical methods well known to the person skilled in the art (see, for example, the esterification process in acid medium described by Fraenkel-Conrat H. and Olcott H. S. 1945, 3. Biol. Chem. 161, 259-269). It is preferably carried out with at least one carboxyl group of the protein; it is advantageously carried out in acid medium using an alcohol (or an activated derivative of this alcohol obtained according to the classical methods well known to the person skilled in the art).

For example, it is suggested to use an aliphatic alcohol containing between 1 and 5 carbon atoms (methanol, ethanol, etc.).

The conditions under which the esterification reaction is carried out (temperature, duration, pressure, acid concentration, etc.) are a function of the desired degree of esterification and, more generally, of the final products that one wishes to obtain.

The esterified protein is then subjected to the action of at least one proteolytic enzyme under the usual conditions of the relevant technical field in order to cleave the amino acid chain (see, for example, the work edited by A. Neuberger and K. Brocklehurst (1987) in the series New Comprehensive Biochemistry, Vol. 16: Hydrolytic Enzymes, Elsevier, or the work edited by R. J. Beynon and J. S. Bond (1989) in the series The Practical Approach Series: Proteolytic enzymes, a practical approach, IRL PRESS).

Any proteolytic and/or peptidase enzyme (protease, peptidase) can be used in this step (pepsin, papain, trypsin, chymosin, chymotrypsin, thermolysin, etc.); the hydrolysis can be carried out with one of these enzymes or with several of them, sequentially or simultaneously.

According to a variant of the process of the invention, the esterification reaction is preceded by a partial hydrolysis of the protein by means of one or more proteolytic or peptidase enzymes. The degree of partial hydrolysis is left to the judgment of the person skilled in the art, who has a wide margin of discretion depending on the products he wishes to obtain. The only restriction is not to obtain total hydrolysis of the protein or peptide.

Surprisingly, the fact that the hydrolysis process is preceded by an esterification reaction allows the sensitivity of the protein to enzymatic hydrolysis to be increased or modified. The enzyme is "baited" by the modification of the protein; depending on the products used and the reaction conditions, new cleavage sites can be obtained and/or certain common cleavage sites can be suppressed with respect to the native protein. This approach makes it possible to obtain atypical cleavage sites, leading to the production of new peptides, at least some of which are esterified.

Carrying out the process according to the invention results in a mixture of peptides or amino acids and their esters. These peptides, amino acids and corresponding esters can then be separated and purified to obtain homogeneous populations.

Esterification by an aliphatic alcohol results in a protein esterified at its C-terminal carboxyl group and at the pendant carboxyl groups of some aspartyl and/or glutamyl residues. The subsequent action of the proteolytic enzyme makes it possible to obtain a terminal peptide whose C-terminal carboxyl group is esterified; all the other peptides obtained, esterified or not, contain a non-esterified C-terminal carboxyl group.

This substitution on the carboxy group makes it possible to suppress a negative charge on the protein or peptide obtained (charge suppression above the pK value of the COOH group of the constitutive aspartic and glutamic acid residues). It increases the isoelectric point of the protein or peptide; it makes them more basic and more hydrophobic, which leads to a better affinity and interaction with the mostly negatively charged biological and artificial interfaces.

According to the invention, the treatment of amino acid residues by esterification followed by proteolysis enables the preparation of new proteins or peptide populations. The means for carrying it out are simple, inexpensive, non-toxic and preserve most of the nutritional values of the protein.

The obtained esterified peptides have different electrostatic properties, hydrophobicity and amphiphilicity, which gives them special interfacial, physiological, biological and immunological properties.

Esterification can also be a simple means of increasing the sensitivity of a structured protein to enzymatic action.

There are various fields of application of the invention; the products obtained can be used advantageously as ingredients, additives or active ingredients in food preparations, pharmaceutical or cosmetic preparations.

Such applications are similar to those already described for the enzymatic hydrolyses of known proteins, as mentioned in the bibliographical references cited at the beginning of the description and to illustrate the state of the art.

Examples Example 1: Pepsin proteolysis of β-lactoalobulin

Bovine β-lactoglobulin (variant B) is prepared according to the method of Mailliart P. and Ribadeau-Dumas B. (1988, J. Food Sci. 53, 743-745).

Esterification

The esters of β-lactoglobulin are prepared using a procedure derived from that described by Fraenkel-Conrat H. and Olcott H. S. (1945, J. Biol. Chem. 161, 259-268).

The β-lactoglobulin is suspended in ethanol to obtain a 2% suspension. 12 N hydrochloric acid is carefully added while stirring to obtain a protein-alcohol suspension with an acidity of 0.06-0.68 N. This preparation is stirred at 4ºC for several days, depending on the degree of esterification desired. After drying in vacuum, the samples are stored at a temperature of -80ºC. A control sample is prepared in the same way but without the addition of HCl.

Analysis of esterified proteins

To determine the degree of esterification of β-lactoglobulin with ethanol, the color reaction with hydroxylammonium chloride developed by Halpin M. I. and Richardson T. (1945, J. Dairy Sci. 68, 3189-3198) was used, modified according to Bertrand-Harb C., Chobert J. M., Dufour E., Haertle T. (1991, Sci. Aliments 11, 641-652).

In the present case, the β-lactoglobulin was 40% esterified.

hydrolysis

The β-lactoglobulin and esterified β-lactoglobulin samples were subsequently hydrolyzed with pepsin (porcine pepsin: 3200- 4500 BAEE U/mg from Sigma Chimie, St-Quentin Fallavier, France).

Pepsin (1 mg per ml H₂O, initial concentration) is added at an enzyme/protein substrate ratio (E/S) of 2%. The mixture is incubated at 37ºC. Samples are taken after one, two, four, twenty and forty hours; hydrolysis is stopped by adding 1.5 volumes of a 0.2 M Tris-HCl buffer, pH 8.0.

Results

The various peptides obtained were purified and identified based on their amino acid composition and their N-terminal sequence.

The HPLC chromatographic profile of the pepsin hydrolysate (after 40 hours of hydrolysis) of the ethylated β-lactoglobulin is shown in Fig. 1 (C₁₈ column, porosity 10 µm, length 25 cm, inner diameter 0.4 cm, from SFCC Shandon, Gagny, France).

The primary structure of β-lactoglobulin B with the pepsin cleavage sites (*) is shown in Fig. 2.

Fig. 3 shows in tabular form the amino acid composition and the N-terminal sequences of the pepsin peptides of the ethylated β-lactoglobulin (ester).

A kinetic study of the enzymatic action on an esterified derivative of β-lactoglobulin shows that the esterified protein is very rapidly hydrolyzed in aqueous solution, while the native protein is insensitive to such proteolysis.

The invention enables the hydrolysis of this protein, which would not be hydrolyzed without the described modification; on the other hand, it leads to the creation of cleavage sites that are not usual for the enzyme used.

For ethylated β-lactoglobulin, 31 cleavage sites were identified: Gln5-Thr6; Thr6-Met7; Leu10-Asp11; Asp11-Ile12; Gln13-Lys14; Trp19- Tyr20; Leu22-Ala23; Ser27-Asp28; Asp28-Ile29; Leu32-Asp33; Leu39- Arg40; Val41-Tyr42; Leu46-Lys47; Ile56-Leu57; Leu57-Leu58; Trp61- Glu62; Lys75-Thr76; Val81-Phe82; Phe82-Lys83; Leu87-Asn88; Glu89- Asn90; Val92-Leu93; Leu93-Val94; Asp98-Tyr99; Met107-Glu108; Leu117-Ala118; Asp130-Glu131; Leu133-Glu134; Phe136-Asp137; Asp137-Lys138; Leu149-Ser150.

Example 2: Pepsin hydrolysis of β-casein

Crude β-casein A1 is prepared according to the method described by Zittle C. A. and Custer J. H. (1963, J. Dairy Sci. 46, 1183-1188). The substrate is then purified by chromatography according to the method of Mercier J. C., Maubois J. L., Poznanski S., Ribadeau-Dumas B. (1968, Bull. Soc. Chim. Biol. 50, 521-530) on a Q-Sepharose Fast Flow column (registered trademark) from Pharmacia, Uppsala, Sweden.

Example 2a Esterification

The esterified β-casein is prepared using a modification of the procedure described by Fraenkel-Conrat and Olcott (1945, loc. cit.). The purified β-casein is dispersed in ethanol to obtain a 2% suspension. 12 N hydrochloric acid is carefully added to the protein-alcohol suspension while stirring to obtain a suspension with an acidity of 0.06-0.68 N. The product obtained is stirred at 4ºC for several days, depending on the degree of esterification desired. After drying in vacuo, the samples are stored at a temperature of -80ºC. A control sample is prepared in the same way but without hydrochloric acid.

Analysis of esterified proteins

The degree of esterification of β-casein with ethanol was determined using the color reaction with hydroxylammonium chloride developed by Halpin and Richardson (1985) and modified by Bertrand-Harb et al. (1991).

In the present case, the β-casein was esterified to 55%.

hydrolysis

The β-casein and the esterified β-casein (2 mg/ml) were dissolved in 20 mM citric acid, pH 2.6. Pepsin (1 mg per ml H₂O, initial concentration) was added at an enzyme/protein substrate ratio (E/S) of 0.2%. The resulting mixture was incubated at 20ºC. Samples were taken after 1, 2, 4, 20 and 40 hours; hydrolysis was by addition of 1.5 volumes of a 0.2 M Tris-HCl buffer, pH 8.0.

Example 2b

A similar esterification was carried out with β-casein using methanol instead of ethanol; the subsequent hydrolysis of the methylated β-casein was carried out in the same manner as the hydrolysis of the ethylated β-casein described above. In this example too, the β-casein was esterified to 55%.

Results

Fig. 4 shows the HPLC chromatographic profile of a pepsin hydrolysate of native β-casein after 10 hours of hydrolysis.

Figure 5 shows the HPLC chromatographic profile (after 10 hours hydrolysis) of a pepsin hydrolysate of methylated β-casein (ester) prepared according to Example 2b.

Figure 6 shows the HPLC chromatographic profile (after 10 hours hydrolysis) of a pepsin hydrolysate of ethylated β-casein (ester) prepared according to Example 2a.

Fig. 7 shows the primary structure of β-casein A1 with the pepsin cleavage sites within the native β-casein (*), within the methylated β-casein (+) and within the ethylated β-casein (o).

Figures 8, 9 and 10 show in tabular form the amino acid compositions and the N-terminal sequences of the pepsin peptides of native β-casein, methylated β-casein (ester) or ethylated β-casein (ester).

In the case of esterified β-casein, six new cleavage sites were identified: Glu11-Ile12; Asn73-Ile74; Met156-Phe157; Val162-Leu163; Leu198-Gly199; Ile207-Ile208.

Example 3: Trypsin hydrolysis of β-lactoglobulin

The β-lactoglobulin and the esterified β-lactoglobulin are prepared according to Example 1 and hydrolyzed with trypsin (TPCK-treated bovine trypsin, 10,000-13,000 U/mg; from Sigma Chimie).

The β-lactoglobulin and the esterified β-lactoglobulin (2 mg/ml) are dissolved in a 0.2 M Tris-HCl buffer at pH 8.0. The trypsin, previously dissolved in 0.01 N HCl, is added at an enzyme/protein substrate ratio (E/S) of 2.5%. The resulting mixture is incubated at 37ºC and the hydrolysis is stopped after 24 hours by adding 0.5 volumes of 0.2 N HCl.

Results

Figure 11 shows the HPLC chromatographic profile of a trypsin hydrolysate of native β-lactoglobulin (after 24 hours of hydrolysis).

Fig. 12 shows the HPLC chromatographic profile (after 24 hours hydrolysis) of a trypsin hydrolysate of ethylated β-lactoglobulin (ester).

Fig. 13 shows the primary structure of β-lactoglobulin B; indicated are the 16 trypsin peptides obtained and the atypical cleavage site (*) of the esterified β-lactoglobulin.

Figures 14a, 14b and 14c show in tabular form the amino acid composition and the N-terminal sequences of the trypsin peptides of the native β-lactoglobulin and the ethylated β-lactoglobulin.

Analysis of the trypsin peptides of β-lactoglobulin shows that esterification does not prevent any cleavage of a target bond, even if an esterified aspartyl or glutamyl residue is in the vicinity of the target bond, resulting in the recovery of esterified peptides. An atypical cleavage site was detected: Met145-His146.

Example 4: Trypsin hydrolysis of β-casein

The β-casein and the esterified β-casein obtained according to Example 2 were subjected to the action of trypsin (TPCK-treated bovine trypsin, 10,000-13,000 U/mg).

The native β-casein and the esterified β-casein (2 mg/ml) are dissolved in a 0.2 M Tris-HCl buffer at pH 8.0. The trypsin, previously dissolved in 0.01 N HCl, is added at an enzyme/protein substrate ratio (E/S) of 1.25º10. The resulting mixture is incubated at 20ºC; hydrolysis is stopped after 24 hours by adding 0.5 volumes of 0.2 N HCl.

Results

Fig. 15 shows the HPLC chromatographic profile of a trypsin hydrolysate of native β-casein (after 24 hours of hydrolysis).

Fig. 16 shows the HPLC chromatographic profile (after 24 hours hydrolysis) of a trypsin hydrolysate of methylated β-casein (ester).

Fig. 17 shows the HPLC chromatographic profile (after 24 hours hydrolysis) of a trypsin hydrolysate of ethylated β-casein (ester).

Fig. 18 shows the primary structure of β-casein A1; the letters A to N indicate the trypsin peptides of the native β-casein, and the atypical cleavage sites have been indicated: for the ethylated β-casein (o) and for the methylated β-casein (*).

Figures 19a and 19b show in tabular form the amino acid composition and the N-terminal sequences of the trypsin peptides of the native β-casein, the methylated β-casein and the ethylated β-casein.

As with β-lactoglobulin, analysis of the trypsin peptides of β-casein shows that esterification does not prevent any cleavage of a target bond, even when an esterified aspartyl or glutamyl residue is in the vicinity of the target bond, resulting in the recovery of esterified peptides. Atypical cleavage sites were identified: Phe52-Ala53; Gln79-Thr80; Ser122-Gln123; Ser124-Leu125; Phe190-Leu191; Tyr193-Gln194; Val197-Leu198.

Claims (7)

1. Process for treating a substrate of animal or plant origin containing at least one protein or peptide provided with at least one esterifiable group other than the C-terminal carboxy group of the chain, by an enzymatic hydrolysis technique leading to the obtaining of peptide products, characterized in that it consists in subjecting, before the enzymatic hydrolysis operation, the protein substrate to an esterification reaction with at least one esterifiable group of the protein or peptide other than the possible esterification of the C-terminal carboxy group of the chain, the sequence of esterification and hydrolysis operations making it possible to obtain peptides at least some of which are esterified on at least one of their esterifiable groups, with the exception of their C-terminal carboxy group. 2. Process according to claim 1, characterized in that it consists in carrying out the esterification reaction with at least one carboxyl group of the protein or peptide. 3. Process according to claim 2, characterized in that the esterification of the protein or peptide takes place in the presence of an alcohol. 4. Process according to claim 3, characterized in that the esterification takes place in the presence of an aliphatic alcohol containing between 1 and 5 carbon atoms. 5. Process according to one of claims 1 to 4, characterized in that the esterification reaction is preceded by a partial hydrolysis of the protein. 6. Process according to one of claims 1 to 5, characterized in that the esterification reaction is followed by a sequence of hydrolysis operations using various proteolytic or peptidase enzymes. 7. Process according to one of claims 1 to 6, characterized in that a protein substrate is used which originates from milk and which contains in particular β-casein or β-lactoglobulin.